Chromosomal aberrations play a central role in the pathogenesis of many human malignant diseases, including hematologic neoplasms such as lymphoma and leukemia. Chromosomal abnormalities, characterized by structural changes or defects in one or more chromosomes, generally involve translocation, wherein a chromosome fragment is switched between non-homologous chromosomes; inversion, wherein the nucleotide sequence of a chromosome fragment is reversed; deletion (loss of a chromosomal fragment); insertion (incorporation of genetic material); duplication (repetition of an individual chromosome segment); or ring formation. These acquired genetic anomalies usually result in either activation of a quiescent gene or creation of a hybrid gene encoding a chimeric fusion oncoprotein, which triggers the malignant transformation. The chimeric fusion proteins created by cancer-associated chromosomal anomalies are ideal therapeutic targets because they are unique to the disease; they only exist in the malignant cells, not in the patient's normal cells (Cobaleda, C. et al., Bioassays (1995) 23:922).
A number of therapeutic agents which target expression of chimeric fusion genes are known in the art, including zinc-finger proteins (Choo, Y., et al., Nature (1994) 372:642), hammerhead-based ribozymes (James, H. A, and I. Gibson, Blood (1998) 91:371), and antisense RNA (Skorski, T. et al., Proc. Natl. Acad. Sci. USA (1994) 91:4504–4508). Each of these agents have inherent limitations. Zinc-finger proteins act at the DNA level, interacting with the target sequence and blocking transcription. However, gene fusions occur randomly and within introns, hence requiring a unique or “custom” zinc-finger for each patient. Antisense approaches, using either single-stranded RNA or DNA, act in a 1:1 stoichiometric relationship and thus have low efficacy, as well as questionable specificity (Skorski et al., supra). Hammerhead ribozymes, which because of their catalytic activity can degrade a higher number of target molecules, have been used to overcome the stoichiometry problem associated with antisense RNA. However, hammerhead ribozymes require specific nucleotide sequences in the target gene, which are not always present.
More recently, double-stranded RNA molecules (dsRNA) have been shown to block gene expression in a highly conserved regulatory mechanism known as RNA interference (RNAi). Briefly, the RNAse III Dicer processes dsRNA into small interfering RNAs (siRNA) of approximately 22 nucleotides, which serve as guide sequences to induce target-specific mRNA cleavage by an RNA-induced silencing complex RISC (Hammond, S. M. , et al., Nature (2000) 404:293–296). In other words, RNAi involves a catalytic-type reaction whereby new siRNAs are generated through successive cleavage of long dsRNA. Thus, unlike antisense, RNAi degrades target RNA in a non-stoichiometric manner. When administered to a cell or organism, exogenous dsRNA has been shown to direct the sequence-specific degradation of endogenous messenger RNA (mRNA) through RNAi. WO 99/32619 (Fire et al.) discloses the use of a dsRNA of at least 25 nucleotides in length to inhibit the expression of a target gene in C. elegans. Sharp, P. A. , Genes & Dev. (2001) 15:485–490, suggests that dsRNA from a related but not identical gene (i.e.,>90% homologous) can be used for gene silencing if the dsRNA and target gene share segments of identical and uninterrupted senquences of significant length, i.e., more than 30–35 nucleotides. Unfortunately, the use of long dsRNAs in mammalian cells to elicit RNAi is usually not practical, due to the deleterious effects of the interferon response, as well as the inherent difficulties in delivering large molecules into a cell.
WO 00/44895 (Limmer, 2000) discloses the use of short dsRNA of less than 25 nucleotides (siRNA) for inhibiting expression of target genes in vertebrate cells. Similarly, WO 01/75164 A2 (Tuschl et al., 2001) discloses dsRNA of about 21 to 23 nucleotides for use in gene silencing by RNAi. Although the dsRNAs described in these references are small enough for intracellular delivery, neither reference suggests the use of siRNAs for inhibiting the expression of a chimeric fusion gene. Moreover, given the fact that chimeric fusion genes contain sequences from the cellular genes from which they originate, one would anticipate problems with specificity of inhibition, i.e., inhibition of both the chimeric fusion gene and the cellular genes. According to Sijen, T., et al., Cell (2001) 107:465–476, and Lipardi, C., et al., Cell (2001) 107:297–307, one strand of the siRNA would be elongated into a region that is complementary to the cellular genes. The new siRNAs formed by subsequent cleavage of the elongated products would have sequences that correspond exclusively to the cellular gene. Thus, one would anticipate inhibition of expression of the target gene as well as the cellular genes.
Finally, Cobaleda, I. and I. Sanchez-Garcia, Blood (2000) 95(3):731–737, discloses the use of a sequence-specific catalytic RNA subunit of RNase P from E. coli (MI RNA) to cleave target mRNA corresponding to the junction site in a bcr-abl fusion gene. However, the MI RNA approach suffers from the same deficiencies as the antisense approach, namely the potential for an interferon response and the inherent difficulties in delivering large molecules to cells. Moreover, because of its large size, production of therapeutic or commercial amounts of MI RNA cannot reasonably be accomplished using solid-phase synthesis. Instead, MI RNA must be prepared through enzymatic synthesis, which is costly.
Thus, despite significant advances in the field, there remains a need for agents that target expression of chimeric fusion genes associated with chromosomal aberrations. In particular, agents that are small enough for efficient intracellular delivery, and which have both high efficacy (hence are effective at low dosages) and high specificity for the target fusion gene would be therapeutically beneficial. Such agents would be useful for treating diseases caused by chromosomal anomalies, particularly malignant diseases such as lymphoma and leukemia.